Epstein-Barr virus (EBV) causes infectious mononucleosis and is an important cause of human B-cell and epithelial cell cancers. The switch between latent and lytic infection is mediated by two viral immediate-early (IE) proteins, BZLF1 (Z) and BRLF1 (R). There are two types of EBV, type 1 (T1) and type 2 (T2), but relatively little is known about T2 EBV. T2 EBV is impaired for the ability to transform B cells in vitro due to decreased expression of an EBV oncoprotein, LMP1. However, our preliminary studies demonstrate that T2 EBV induces B-cell lymphomas in a humanized mouse model that are highly lytic, and is also more lytic in oral epithelial cells. Thus, we hypothesize that enhanced lytic infection is a major phenotype of T2 EBV. Our analysis of publically available EBV genomes indicates that all T2 EBV share the same variant (Zp-V3) of the viral promoter (Zp) that governs whether EBV infection is latent or lytic in B cells, and also contain the same variants of the Z and R IE proteins. The T2-encoded Z protein contains 9 amino acid (aa) differences compared to Z encoded by T1 viruses, all located within functionally important regions of the 245 aa protein. Neither the functions of the T1 versus T2 forms of Z, nor the activities of theT1 versus T2 forms of the Z promoter, have been compared. Both the T2 form of the Z promoter, and the T2 form of the Z protein, have been reported to be over-represented in certain types of EBV-infected cancers relative to their frequency in non-malignant samples, and we have recently discovered that T1/T2 hybrid viruses (containing the T2 form of the Z/R IE locus within otherwise T1 EBV viruses) are over- represented in EBV isolated from Burkitt lymphomas (BLs). We have also discovered that the T2, but not T1, form of the Z promoter confers enhanced lytic EBV reactivation in antigen-stimulated B cells due to its ability to bind the NFATC1 cellular transcription factor, and our preliminary results indicate that the T2 Z/R proteins also have an enhanced ability to induce lytic reactivation in B cells. We hypothesize that T2 EBV strains are much more lytic than T1 EBV strains due to differences in the Z promoter, the Z and/or R IE proteins, and decreased LMP1. We also hypothesize that hybrid T1/T2 EBV strains (containing the T2 form of the Z/R IE locus) have an increased ability to enter lytic infection relative to pure T1 strains, and that this difference enhances their malignant potential. Our Specific Aims are to 1) use a humanized mouse model to define EBV genes contributing to the enhanced lytic phenotype of T2 EBV infection in B cells, and to determine if a hybrid T1/T2 EBV virus resembling hybrid viruses found in human BLs is more lytic, or more transforming, than pure T1 EBV; 2) compare the phenotypes of T1, T2, and T1/T2 hybrid viruses in undifferentiated and differentiated oral epithelial cells, and 3) compare the functions of the T1 versus T2 Z and R proteins, and T1 versus T2 Z and R promoters, in vitro in B cell and epithelial cell models, and define mechanism(s) for any differences. The proposed studies should expand our understanding of T2 EBV, and may reveal why T1/T2 hybrid EBV strains are over-represented in cancers.